We propose to create a flexible polycistronic system for expressing multiple engineered receptors to facilitate the engineering of tissues, such as cardiac cells, brain, and bone. We developed new class of G-protein- coupled receptors (GPCRs) that are called RASSLs (Receptors Activated Solely by a Synthetic Ligand). RASSLs are engineered to be unresponsive to endogenous hormones, yet still activated by small-molecule drugs with nanomolar affinities and relatively few side effects in vivo, making them ideally suited for tissue- engineering studies. Our prototype RASSL (RO1) activates the Gi signaling pathway, inhibiting cAMP formation. When RO1 is expressed in specific tissues, it can affect diverse physiological processes, such as heart rate, remodeling of heart, neurotransmission, and bone growth. We have helped to establish a growing community of researchers who have engineered new RASSLs to activate all the major GPCR pathways, including Gs, which increases cAMP formation, and Gq, which stimulates calcium mobilization. We now propose to tackle the next major challenge for RASSL users. The lack of robust and controllable in vivo delivery systems for individual or multiple RASSLs has hindered wider use of RASSLs by the scientific community. To meet this need, we will develop a flexible system for expressing multiple RASSLs, using a set of common components that will mimic any GPCR signaling combination with these specific aims. Aim 1. To create an in vivo expression system for optimal spatial and temporal control of RASSL expression. Our ultimate goal is to combine Cre and Tet systems for RASSL expression. We will evaluate each strategy independently in a series of three vectors. We will use the Gi-RASSL in the cardiac pacemaker as a model system with robust in vivo responses. Aim 2. To characterize the physiological responses to each major RASSL-induced GPCR-signaling pathway in mouse cardiac pacemaker tissue and ES cells. Gs-, Gi-, and Gq-RASSLs with high and low levels of basal signaling will be expressed in cardiac pacemaker tissue. To define RASSL-induced phenotypes, we use a cell-culture model of ES cell-derived contracting myocytes, as well as developing pacemaker tissue in mouse embryos and cardiac monitoring in adult mice. Aim 3. To determine the physiological effects of co-stimulating multiple GPCR signaling pathways. We will use the 2A polycistronic system to co-express combinations of RASSLs that activate each of the three major signaling pathways. The four signaling combinations in this series will include Gs-Gi, Gs-Gq, Gi-Gq, and Gs-Gi-Gq. We will also use RASSLs that can be activated by a single ligand, so as to insure co-stimulation. Public Health Relevance Statement (Provided by Applicant): The fundamental importance of this project to human health is to provide powerful new tools for tissue engineers, who are working to restore function to many tissues, such as the heart, brain and bone. RASSLs also provide basic insights into the actions of GPCRs that are the targets of many widely used pharmaceuticals. Finally we are using the cardiac pacemaker as a model system. We hope to gain fundamental insights into cardiac arrhythmias that are a major cause of morbidity and mortality.